【作者】 孙昆峰；

【导师】 汪铭书； 程安春；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2013， 博士

【摘要】 作为疱疹病毒目(Herpesvirales)疱疹病毒科(Herpesvirales)α-疱疹病毒亚科(Alphaherpesvirinae)马立克病毒属(Mardivirus)的一员,鸭瘟病毒(Duck plague virus, DPV)是一种泛嗜性全身性感染病毒,主要引起鸭、鹅、天鹅等雁形目水禽的急性接触性传染病——鸭瘟。该病以血管损伤、组织出血、消化道黏膜糜烂、淋巴器官出现病变及实质器官的退行性变化为特征,是影响水禽养殖业的重要疫病。目前,主要依靠接种灭活疫苗和鸡胚化弱毒苗来预防鸭瘟的发生。灭活疫苗安全性好,但免疫维持期短、剂量较大、成本较高；鸡胚化弱毒苗虽然免疫效果较好,但是存在毒力返强和隐性带毒等安全隐患,而且现有的诊断方法无法区别自然感染野毒和弱毒疫苗免疫的动物。因此,研制更为安全有效的新型基因工程疫苗具有重要的理论和应用价值。生物信息学分析表明鸭瘟病毒糖蛋白C (glycoprotein C, gC)在不同毒株间高度保守,原核表达结果显示gC具有良好的免疫原性。因此,本研究选择gC基因作为保护性抗原基因,将其克隆至真核表达载体pcDNA3.1(+)以构建鸭瘟病毒gC基因疫苗。酶切分析和间接免疫荧光法证实gC基因正确插入并能够在COS7细胞中表达。为了大量获取构建的真核表达质粒,本研究初步摸索了该质粒高密度发酵的条件,随后进行了质粒大抽和纯化,分光光度计、酶切分析和琼脂糖电泳结果表明获得的质粒杂质少、超螺旋结构含量达90%以上。为了阐明免疫佐剂及免疫途径对鸭瘟病毒gC基因疫苗的影响,本研究应用gC特异的基于TaqMan探针的定量PCR分析脂质体/gC基因疫苗复合物及壳聚糖/gC基因疫苗复合物通过肌注、口服、滴鼻等途径进入鸭体内后在各组织器官的分布规律,同时与基因枪轰击和肌注裸质粒不同剂量进行比较。试验结果表明脂质体和壳聚糖能够有效提高鸭瘟病毒gC基因疫苗在鸭体内的分布,而基因枪轰击的转染效率最高,口服有利于基因疫苗在消化道和法氏囊的分布,滴鼻能提高基因疫苗在呼吸道的分布,肌肉注射能使基因疫苗更快速地分布到各组织;另外,肌肉注射和基因枪轰击法均与免疫剂量呈现一定的相关性。当前3株已经完成全基因测序的鸭瘟病毒,CHv株基因组最大,由162175碱基对构成。作为一种强毒株,对其进行基因重组研究,能够更有效地揭示基因在鸭瘟病毒中的作用。本研究首先以CHv gC基因为靶位点,克隆了该基因两侧各1.3kb左右的片段作为同源臂,将增强绿色荧光蛋白(enhanced green fluorescent protein, EGFP)基因插入其中,构建了被EGFP替换了gC基因前1246bp的DPV重组转移载体pUC-△gC-EGFP。为了有效控制EGFP基因的插入方向以使其表达不受gC启动子影响,本研究首先删除了pEGFP-C1上Xho Ⅰ和Xba Ⅰ(不包括二者)之间的多克隆位点,接着通过PCR的方式扩增出包含CMV立即早期启动子、EGFP基因、转录终止信号SV40polyA共1579bp的片段,再将其克隆至T载体。随后,将pUC-△gC-EGFP质粒和DPV CHv共同转染鸭胚成纤维细胞(duck embryo fibroblast, DEF),倒置荧光显微镜下结合绿色荧光进行空斑纯化,成功获得了纯化的表达EGFP基因的DPVgC缺失株DPV-△gC-EGFP.将DPV-△gC-EGFP在DEF上连续传30代以上,并进行PCR检测、测序分析及其在鸭体内、体外生物学特性研究。DPV-△gC-EGFP在DEF上连续传代表明EGFP基因能够稳定遗传并正确表达。PCR和测序结果显示DPV-△gC-EGFP中gC片段内部序列与预期一致。与DPV亲本株相比,DPV-△gC-EGFP病毒总的滴度下降了约50倍,受影响最大的是病毒释放到上清中的滴度下降了40倍；MEM液体培养基中可见DPV-△gC-EGFP主要通过细胞间传递；透射电镜下,很难发现游离的DPV-△gC-EGFP病毒颗粒；这些结果表明gC在病毒的装配中发挥作用。另外,可以观察到感染DPV-△gC-EGFP的DEF形成了多核巨细胞病毒,说明gC在鸭瘟病毒形成的细胞融合中发挥抑制作用。接种鸭的试验表明DPV-△gC-EGFP致病性降低,能诱发一定的中和抗体,并为接种鸭提供100%抗强毒攻击的保护,揭示DPV gC缺失株有望发展成为有效预防鸭瘟的基因工程疫苗。鸭瘟病毒糖蛋白E (glycoprotein E, gE)基因在不同毒株间相似性为99%,因为具有较好的免疫原性和免疫反应性,所以已经建立了基于gE的ELISA方法。鉴于在α-疱疹病毒中gE是重要的毒力基因,本研究构建了表达EGFP的鸭瘟病毒gE缺失株DPV-△gE-EGFP。PCR、测序分析及免疫荧光法结果表明DPV-△gE-EGFP中gE基因被EGFP基因替换,不再表达gE蛋白。连续传代结果说明DPV-△gE-EGFP能够稳定遗传并表达EGFP蛋白。与亲本株相比,DPV-△gE-EGFP病毒滴度降低约9倍,形成空斑能力显著降低,一步生长曲线表明病毒感染前期增殖减慢,这些结果表明gE的缺失影响了病毒在细胞间传递的能力。动物试验方面,DPV-△gE-EGFP除了引起接种鸭轻微的体温升高,未表现其它异常症状,说明gE基因是鸭瘟病毒重要的毒力基因。从中和抗体试验结果来看,虽然与gC缺失株相比显著降低,但是DPV-△gE-EGFP同样可为接种鸭提供100%抗强毒攻击的能力,并且接种强毒后,无异常临床变化,揭示DPV gE缺失株更有希望发展成为一种有效预防鸭瘟的标记疫苗。更多还原

【Abstract】 As one virus of genus Mardivirus, subfamily Alphaherpesvirinae, family Herpesviridae, order Herpesvirales, duck plague virus (DPV), a pan-tropic systemic infection virus, is the cause of an acute contagious disease-duck plague-of ducks, geese, and swans, which is characterized by vascular damage, tissue hemorrhages, digestive mucosal eruptions, lesions of lymphoid organs, and degenerative changes in parenchymatous organs. The disease is an important epidemic affecting waterfowl breeding industryAt present, the prevention and control of duck plague is mainly depended on both live attenuated and inactivated vaccines. Inactivated vaccines are relative safety, but give only short-term immunity, and need larger doses and higher costs. Although high immunogenicity and longer term of immunoprotection do live attenuated vaccines possess, they still have some problem need to solve, including the latent dangers of persistence infection and virulence reversion, and the difficulty in distinguishing vaccinated birds from infected ones. Therefore, it has important practical significance to develop more safe and effective genetically engineered vaccines.Bioinformatic analysis showed that glycoprotein C of duck plague virus is highly conserved among different strains. And it was proved by prokaryotic expression that glycoprotein C had high immunogenicity. Accordingly, gC gene was chosed as the protective antigen gene and subcloned into the eukaryotic expression vector pcDNA3.1(+) in this study. The results of restriction endonuclease digestion and indirect immunofluorescence assay confirmed that the gC gene was inserted correctly and expressed in COS-7cells. In order to obtain a large number of the eukaryotic expression plasmid, the condition of high-cell-density fermentation was studied. Obtained from maxipreparation and purification, plasmid was analyzed by spectrophotometer, restriction analysis and agarose gel electrophoresis. It was showed that plasmid contained little impurity and the supercoiled structure up to90%.In order to clarify the effect of immunological adjuvant and immune pathways, complexes of gC gene vaccine with lipid or chitosan were inoculated into ducks by intramuscular, oral and intranasal. At the same time, gC gene vaccine was inoculated into ducks with the gene gun bombardment and intramuscular injection of different doses. And then, the tissue distribution characteristics of gC gene vaccine was analyzed by quantitative real-time PCR based TaqManTM probe. The results showed that1) the liposomes and chitosan can effectively improve the tissue distribution of gC gene vaccine in ducks; 2) the gene gun bombardment has the highest transfection efficiency;3) oral vaccination benefits the distribution of gene vaccine in the digestive tract and bursa of Fabricius;4) intranasal immunization can improve the distribution of gene vaccine in respiratory tract and brain;5) intramuscular injection mediate more rapid plasmid distribution in duck tissues;6) intramuscular and gene gun bombardment are dose-related.CHv strain has the largest genome among three strains of duck plague virus which genomes have been sequenced completely. As a virulent strain, CHV strain was choosed to more effectively reveal the role of the gene in duck plague virus by the recombinant studies. In the present study, gC gene was chosed as target gene. Firstly, two fragments flanking gC gene about1.3kb were amplified and subcloned into pUC19. Then, a DPV transfer vector pUC-AgC-EGFP was constructed, which contained an EGFP reporter. In order to effectively control the insertion direction of the EGFP gene to express against gC promoter, multiple cloning sites between Xho I and Xba I pEGFP-C1were first deleted. And then, a1579bp fragment, which contained the sequences of Human cytomegalovirus (CMV), immediate early promoter and SV40early mRNA polyadenylation signal, was amplified and clonee into T vector. PCR fragment, Co-transfected pUC-△gC-EGFP with DPV CHv in DEF, a recombinant DPV, designated as DPV-AgC-EGFP, was successfully obtained after plague purification which combined with green fluorescent under inverted fluorescence microscope.DPV-AgC-EGFP was examined by continuous passage30or more, PCR, sequencing and biological characteristics in vitro and in vivo. It was shown that EGFP gene can be stably inherited and correctly expressed. The results of PCR and sequencing showed that the gC gene sequence of DPV-AgC-EGFP in line with expectations. Compared with DPV parental strain, the total virus titer of DPV-AgC-EGFP decreased by approximately50times, and the supernatant virus titer was mostly affected and decreased40times. Additionally, the DPV-AgC-EGFP mainly passed through the cell in MEM and it is difficult to find free the DPV-AgC-EGFP virus particles under transmission electron microscope. All above results showed that the DPV gC played a role in virus assembly. In addition, the fact that DEF infected with DPV-AgC-EGFP DEF formed multinucleated giant cells showed that gc maybe inhibit cell fusion. Furthermore, the results in vivo showed DPV-AgC-EGFP reduced the pathogenicity, induced neutralizing antibodies and protected completely vaccinated ducks from challenge of DPV virulent. Therefore, the gC-deleted DPV can be developed to an effective genetically engineered vaccine for duck plague.The similarity of gE gene between different DPV strains Duck Plague virus was99%. A gE-based ELISA method has been established for its good immunogenicity and reactivity. Named as DPV-AgE-EGFP, a gE-deleted DPV, which can express EGFP, was constructed since the gE gene of alphaherpesvirus was an important virulence gene. The results of PCR, sequencing and immunofluorescence assay showed that the gE gene of DPV-△gE-EGFP was replaced by EGFP gene and cannot be expressed no longer. The results of continuous passage showed that the DPV-△gE-EGFP was able to be stably inherited and express EGFP protein. Compared with the DPV parental strain, the virus titer of DPV-AgE-EGFP reduced by about9times, the plaque formed by DPV-AgE-EGFP also decreased, and it was shown by one step growth curves that the proliferation of DPV-AgE-EGFP slowed down in early infection. These results indicated that the deletion of gE impacted the ability of DPV through cell-to-cell spread. Moreover, The results obtained of in vivo studies revealed that the gE gene play an important role in virulence of DPV because there is no abnormal phenomenon in clinical symptoms except slightly increasing in temperature in ducks vaccinated with DPV-△gE-EGFP. Although the neutralizing antibodies induced by DPV-△gE-EGFP were significantly lower than that induced by DPV-AgC-EGFP, the gE-deleted mutant induced completely protection in inoculated ducks from manifestation of clinical symptoms after viral challenge. The results demonstrate that the gE-deletion mutant is good candidate for DPV marker vaccine.更多还原